Novel recombinant antibody specifically binding to b cell antigen, and use thereof

Abstract

The present invention relates to: a recombinant polypeptide specifically binding to B cells; and a composition for preventing or treating B cell lymphoma, comprising same as an active ingredient. The present invention can significantly improve direct killing activity against tumor cells by conjugating concanavalin A, which is a ligand of MPZL1, to an antibody molecule that recognizes a B cell-specific antigen such as CD20. Therefore, unlike therapeutic antibodies having main mechanisms of antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADP), the recombinant polypeptide of the present invention does not depend on operating mechanisms of patient immune systems, and thus can be effectively used in an effective therapeutic composition even for cancer patients with reduced immune activity.

Description

Novel recombinant antibodies specifically binding to B-cell antigens and uses thereof

[0001] The present invention relates to a novel recombinant antibody having significantly enhanced direct cell killing (DCD) activity against B-cell lymphoma.

[0002]

[0003] CD20 is a B cell-specific antigen that is expressed in high concentrations only on the surface of normal and tumor cells of the B lymphocyte lineage and functions as a receptor during B cell activation. It is a hydrophobic protein containing four transmembrane domains, with both the N-terminus and C-terminus located within the cytoplasm.

[0004] The CD20 antigen is not detected at detectable levels on hematopoietic stem cells, B cell progenitor cells, normal plasma cells, or other normal tissues, but is expressed in more than 90% of B-cell non-Hodgkin lymphomas (NHL). CD20 is predicted to be involved in the activation process of cell cycle initiation and differentiation and to function as a calcium ion channel. Therefore, anti-CD20 antibodies serve to enhance the host's natural defense mechanisms to attack and kill B cells that bind via the CD20 antigen. This cell death is achieved through the complement-dependent cytotoxicity (CDC) pathway and the antibody-dependent cell-mediated cytotoxicity (ADCC) pathway.

[0005] Currently, commercially available anti-CD20 antibodies include rituximab, a type 1 CD20 antibody, and obinutuzumab, a type 2 CD20 antibody. Type 1 antibodies, such as rituximab, translocate CD20 to the Triton-insoluble membrane fraction of lipid bilayers, whereas type 2 antibodies, such as obinutuzumab, induce homotypic aggregation of CD20 at cell-cell contact sites. Furthermore, while rituximab does not induce directed cell death (DCD), obinutuzumab efficiently induces DCD through its specific binding to CD20. Accordingly, the present inventors produced an anti-CD20-APEX2 fusion antibody capable of proximity labeling and, through this, searched for a target protein involved in DCD, thereby seeking to discover a new pharmacological agent that can exert a synergistic tumor cell killing effect when administered in combination with existing CD20 antibodies or in a fused form with these antibodies.

[0006]

[0007] Numerous papers and patents are referenced and cited throughout this specification. The disclosures of these cited papers and patents are incorporated herein by reference in their entirety to provide a clearer understanding of the state of the art and the scope of the invention.

[0008]

[0009] The present inventors have conducted extensive research to develop an efficient recombinant antibody with improved direct cell death effect while maintaining specific affinity for B-cell antigens including CD20 as a therapeutic antibody for B-cell lymphoma. As a result, they have elucidated for the first time that the MPZL1 (Myelin Protein Zero Like 1) protein is involved in the antibody-induced direct cell death mechanism of B-cell lymphoma, and have discovered that conjugating concanavalin A, a ligand of MPZL1, to a B-cell lymphoma-specific antibody can be used as an efficient anticancer composition with significantly increased cell death activity, thereby completing the present invention.

[0010] Accordingly, the purpose of the present invention is to provide a recombinant polypeptide that specifically binds to B cells and a composition for preventing or treating B cell lymphoma containing the same as an active ingredient.

[0011]

[0012] Other objects and advantages of the present invention will become more apparent from the detailed description, claims and drawings below.

[0013]

[0014] According to one aspect of the present invention, the present invention provides a recombinant polypeptide that specifically binds to a B cell comprising:

[0015] (a) an antibody or antigen-binding fragment thereof that specifically binds to a B-lymphocyte antigen; and

[0016] (b) Concanavalin A protein or a functional fragment thereof bound to (a).

[0017] The present inventors have conducted extensive research to develop an efficient recombinant antibody with improved direct cell death effects while maintaining specific affinity for B-cell antigens including CD20 as a therapeutic antibody for B-cell lymphoma. As a result, we have elucidated for the first time that the MPZL1 (Myelin Protein Zero Like 1) protein is involved in the antibody-induced direct cell death mechanism in B-cell lymphoma, and have discovered that conjugating concanavalin A, a ligand of MPZL1, to a B-cell lymphoma-specific antibody can be used as an effective anticancer composition with significantly increased cell death activity.

[0018] The term “antibody” as used herein refers to a peptide that recognizes and specifically binds to a specific epitope of a B cell antigen, for example, CD20, and includes complete antibody forms as well as antigen-binding fragments (antibody fragments) of full-length antibody molecules. Complete antibodies comprise two full-length light chains and two full-length heavy chains, each light chain being linked to a heavy chain by a disulfide bond. The heavy chain constant regions are of the gamma (γ), mu (μ), alpha (α), delta (δ), and epsilon (ε) types and have subclasses of gamma1 (γ1), gamma2 (γ2), gamma3 (γ3), gamma4 (γ4), alpha1 (α1), and alpha2 (α2). The light chain constant regions are of the kappa (κ) and lambda (λ) types.

[0019] The term “antigen-binding fragment of an antibody” as used herein means a fragment having significant antigen-antibody binding function within a full-length antibody molecule, and includes Fab, F(ab'), F(ab')2, Fv, and nanobody (or sybody).

[0020] Among antibody fragments, Fab has a structure with variable regions of the light and heavy chains, constant regions of the light chain, and the first constant region (CH1) of the heavy chain, and has one antigen-binding site.

[0021] Fab' differs from Fab in that it has a hinge region containing one or more cysteine ​​residues at the C-terminus of the heavy chain CH1 domain. F(ab')2 antibodies are produced when the cysteine ​​residues in the hinge region of Fab' form a disulfide bond. Fv is the smallest antibody fragment that has only a heavy chain variable region and a light chain variable region. A two-chain Fv has a heavy chain variable region and a light chain variable region linked non-covalently, and a single-chain Fv has a heavy chain variable region and a single chain variable region linked covalently, usually through a peptide linker, or directly at the C-terminus, so that it can form a dimer-like structure like a two-chain Fv.

[0022] The term “heavy chain” as used herein encompasses a full-length heavy chain and fragments thereof comprising a variable region domain VH comprising an amino acid sequence having sufficient variable region sequence to confer specificity for an antigen and three constant region domains CH1, CH2 and CH3.

[0023] The term “light chain” as used herein refers to both full-length light chains and fragments thereof comprising a variable region domain VL and a constant region domain CL, which comprise an amino acid sequence having sufficient variable region sequence to confer specificity for an antigen.

[0024] The variable region domains of the heavy and light chains each contain three CDR regions.

[0025] As used herein, the term “CDR (complementarity determining region)” refers to the amino acid sequence of the hypervariable region of the immunoglobulin heavy and light chains. The heavy chain (HCDR1, HCDR2, and HCDR3) and the light chain (LCDR1, LCDR2, and LCDR3) each contain three CDRs, which provide key contact residues for antibody binding to an antigen or epitope.

[0026] The scope of the antibodies or antigen-binding fragments of the present invention includes variants having conservative amino acid substitutions in the CDR regions. In addition, the antibodies or antigen-binding fragments of the present invention may include variants of the amino acid sequence within the range capable of specifically recognizing a particular B-cell antigen, e.g., CD20. For example, additional changes may be made to the amino acid sequence of the antibody to further improve the binding affinity and / or other biological properties of the antibody. Such modifications include, for example, deletions, insertions, and / or substitutions of amino acid sequence residues of the antibody, and are made based on the relative similarity of the amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge, size, etc. Analysis of the size, shape, and type of amino acid side chain substituents reveals that arginine, lysine, and histidine are all positively charged residues; alanine, glycine, and serine have similar sizes; and phenylalanine, tryptophan, and tyrosine have similar shapes. Based on these considerations, arginine, lysine, and histidine; Alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine can be considered biologically functional equivalents.

[0027] Furthermore, amino acid substitutions in proteins that do not alter the overall activity of the molecule are well known in the art (H. Neurath et al., The Proteins, Academic Press, New York, 1979). The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thr / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly.

[0028] Considering the mutations having the above-described biological equivalent activity, the amino acid sequence constituting the antibody of the present invention is interpreted to also include a sequence showing substantial identity with the sequence described in the sequence listing. The substantial identity means a sequence showing at least 61% homology, in one specific example 70% homology, in another specific example 80% homology, and in yet another specific example 90% homology, when the sequence of the present invention and any other sequence are aligned to the greatest extent possible and the aligned sequences are analyzed using an algorithm commonly used in the art. Alignment methods and algorithms for sequence comparison are disclosed in Huang et al. Comp. Appl. BioSci. (1992) 8:155-65 and Pearson et al. Meth. Mol. Biol. (1994) 24:307-31, etc.

[0029] According to a specific embodiment of the present invention, the B cell-specific antigen is at least one antigen selected from the group consisting of CD19, CD20, CD30 and CD79b.

[0030] The term “Concanavalin A (ConA)” in this specification refers to a carbohydrate-binding lectin protein extracted from sword bean, which specifically binds to α-D-mannose and α-D-glucose, which are non-reducing terminals of various sugars, glycoproteins, and glycolipids. Like most lectins, ConA is a homotetramer composed of four identical subunits, each of which consists of 235 amino acid residues with a molecular weight of 26.5 kDa. 2+ or Ca 2+ It binds to metal atoms such as the alkyl group and each subunit has a binding site, so the entire ConA molecule contains a total of four symmetrical binding sites.

[0031] The term “functional portion” as used herein refers to an analog of the full-length protein, which is a fragment in which some amino acid residues or some domains are deleted from the full-length protein, and which retains its original biological activity and function. For example, a functional portion fragment of a concanavalin A protein may be a fragment containing one or two subunits that constitute a full-length concanavalin A tetramer, and more specifically, may be a dimer containing two subunits.

[0032] According to a specific embodiment of the present invention, the concanavalin A protein or a functional fragment thereof binds to the C-terminus of the antibody or an antigen-binding fragment thereof.

[0033] According to a specific embodiment of the present invention, the concanavalin A protein or a functional fragment thereof is bound to the C-terminus of the antibody or an antigen-binding fragment thereof via a linker.

[0034] As used herein, the term "linker" refers to a linker that physically connects two fusion partners (e.g., biological polymers such as peptides) via a covalent bond. The linker may be a non-peptide linker or a peptide linker. In the case of a non-peptide, it may be a multi-functional ligand compound having two or more active functional groups that acts as a linker between two or more molecules by binding to them, and in the case of a peptide linker, it may be composed of three or more amino acids that form a peptide bond with each fusion partner, thereby separating them at an appropriate spatial distance and forming a single molecule.

[0035] More specifically, the linker is a flexible peptide linker represented by (GGGGS)n (n is an integer from 1 to 5). Even more specifically, n is an integer from 2 to 5, even more specifically, an integer from 3 to 5, and most specifically, 4.

[0036] According to a specific embodiment of the present invention, the functional fragment of concanavalin A is a dimer comprising two monomer subunits constituting a concanavalin A tetramer.

[0037] According to a specific embodiment of the present invention, the two monomer subunits of concanavalin A bind to each heavy chain C-terminus of an antibody that specifically binds to the B cell antigen.

[0038] When a functional fragment of concanavalin A, i.e. a homodimer, binds to a B-cell antigen-specific antibody, two monomers can each bind to the C-terminus of the heavy chain constant region of the antibody, as shown in Figure 17.

[0039] According to a specific embodiment of the present invention, the monomer of concanavalin A used in the present invention comprises an amino acid sequence of sequence number 17.

[0040] According to a specific embodiment of the present invention, the B cell specific antigen is CD20.

[0041] More specifically, the antibody that specifically binds to CD20 may be obinutuzumab or rituximab. In this case, the “antigen-binding fragment of the antibody that specifically binds to a B-cell antigen” may be a fragment of a full-length antibody that includes all or part of the six CDRs of HCDR1 to HCDR3 and LCDR1 to LHCDR3 of obinutuzumab or rituximab. The amino acid sequences of rituximab and obinutuzumab can be obtained from various public databases, for example, U.S. Patent Publication No. 2015-0141620 (rituximab) and U.S. Patent Registration Publication No. 11,110,087 (obinutuzumab), respectively.

[0042] When the antibody specifically binding to CD20 used in the present invention is obinutuzumab, the antibody or antigen-binding fragment thereof may include a heavy chain variable region including the HCDR1 region of the first sequence in the sequence listing; the HCDR2 region of the second sequence in the sequence listing; and the HCDR3 region of the third sequence in the sequence listing.

[0043] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof may additionally include a light chain variable region comprising the LCDR1 region of sequence number 4; the LCDR2 region of sequence number 5; and the LCDR3 region of sequence number 6.

[0044] More specifically, the antibody or antigen-binding fragment thereof may include a heavy chain variable region of sequence number 7 and / or a light chain variable region of sequence number 8.

[0045]

[0046] When the antibody specifically binding to CD20 used in the present invention is rituximab, the antibody or an antigen-binding fragment thereof may include a heavy chain variable region including the HCDR1 region of sequence number 9; the HCDR2 region of sequence number 10; and the HCDR3 region of sequence number 11.

[0047] According to a specific embodiment of the present invention, the antibody or antigen-binding fragment thereof may additionally include a light chain variable region comprising an LCDR1 region of sequence listing 12; an LCDR2 region of sequence listing 13; and an LCDR3 region of sequence listing 14.

[0048] More specifically, the antibody or antigen-binding fragment thereof may include a heavy chain variable region of sequence number 15 and / or a light chain variable region of sequence number 16.

[0049] According to a specific embodiment of the present invention, the recombinant polypeptide of the present invention may additionally comprise an IgG1-specific nanobody. In this case, one or two nanobodies may be included, and may bind to the C-terminus of the concanavalin A protein bound to the B-cell antigen-specific antibody, or may be positioned between the B-cell antigen-specific antibody and the concanavalin A protein.

[0050]

[0051] According to another aspect of the present invention, the present invention provides a nucleic acid molecule encoding the recombinant polypeptide of the present invention as described above.

[0052] The term “nucleic acid molecule” as used herein encompasses DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are the basic structural units of nucleic acid molecules, include not only natural nucleotides but also analogues in which sugar or base moieties are modified (Uhlman et al., Chemical Reviews (1990) 90:543-584). The sequence of a nucleic acid molecule encoding a recombinant polypeptide of the present invention may be modified, and the modifications include additions, deletions, or non-conservative or conservative substitutions of nucleotides.

[0053] The nucleic acid molecule of the present invention is also interpreted to include a sequence that exhibits substantial identity to the nucleotide sequence of the present invention. The substantial identity refers to a nucleotide sequence that exhibits at least 80% homology, in one specific example at least 90% homology, and in another specific example at least 95% homology, when the nucleotide sequence of the present invention is aligned with any other sequence to the greatest extent possible and the aligned sequence is analyzed using an algorithm commonly used in the art.

[0054] According to a specific embodiment of the present invention, the nucleic acid molecule used in the present invention may be an mRNA molecule, and more specifically, may be an in vitrotranscribed (IVT) mRNA.

[0055] When mRNA is used as the nucleic acid molecule of the present invention, various modifications may be made, such as changing the length of the poly(A) tail or substituting some adenine bases; modifying the 5'cap; applying one or more modified nucleosides, in order to improve the expression (translation) efficiency of the recombinant polypeptide of the present invention. Modified nucleosides that may be applied include, but are not limited to, N1-methylpseudouridine, pseudouridine, 2-thiouridine, 5-methyluridine, 5-methylcytidine, and 5-methoxyuridine, and any modified nucleoside known in the art to be capable of reducing the immunogenicity of the mRNA molecule may be applied.

[0056] According to another aspect of the present invention, the present invention provides a gene delivery vehicle comprising the nucleic acid molecule of the present invention as described above.

[0057] According to the present invention, the recombinant polypeptide of the present invention may be obtained recombinantly in vitro by expressing a nucleic acid molecule encoding the polypeptide in a host cell, or may be delivered into a patient's body as a pharmacological ingredient itself, thereby producing the recombinant polypeptide of the present invention in the body through the patient's expression system.

[0058] As used herein, the term “express” means artificially introducing a gene using a gene vector to cause a target cell to express an exogenous gene or to increase the natural expression level of an endogenous gene, thereby making the gene replicable as an extrachromosomal element or by completion of chromosomal integration within the target cell. Therefore, the term “expression” has the same meaning as “transformation,” “transfection,” or “transduction.” More specifically, in the present invention, “express” means causing a target cell to artificially express an exogenous gene.

[0059] As used herein, the term "gene carrier" refers to any means of transporting a gene into a cell. Gene transfer is synonymous with transduction of a gene into a cell, and gene transfer at the tissue level is synonymous with spread of a gene. Accordingly, the gene delivery system of the present invention may be described as a gene transduction system and a gene spread system.

[0060] The gene delivery system of the present invention may comprise an expression cassette, which is a polynucleotide structure containing all elements necessary for the autonomous expression of a gene to be introduced. The expression cassette typically comprises a promoter, a transcription termination signal, a ribosome binding site, and a translation termination signal, which are operably linked to the gene. The expression cassette may be in the form of a self-replicating expression vector. As used herein, the term “operably linked” refers to a functional linkage between a nucleic acid expression control sequence, such as a promoter, a signal sequence, or an array of transcription factor binding sites, and another nucleic acid sequence, whereby the expression control sequence controls the transcription and / or translation of the other nucleic acid sequence.

[0061] The recombinant vector system of the present invention can be constructed through various methods known in the art, and specific methods thereof are disclosed, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001).

[0062] The vector of the present invention can typically be constructed as a vector for cloning or as a vector for expression. In addition, the vector of the present invention can be constructed using a prokaryotic cell or a eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and uses a prokaryotic cell as a host, it generally includes a strong promoter capable of promoting transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for initiating translation, and a transcription / translation termination sequence. When E. coli (e.g., HB101, BL21, DH5α, etc.) is used as a host cell, E. The promoter and operator region of the tryptophan biosynthetic pathway of E. coli (Yanofsky, CJ Bacteriol. 158:1018-1024 (1984)) and the left-hand promoter of phage λ (pLλ promoter, Herskowitz, I. et al., Ann. Rev. Genet. 14:399-445 (1980)) can be used as the regulatory region. When Bacillus is used as the host cell, the promoter of the toxin protein gene of Bacillus thuringiensis (Appl. Environ. Microbiol. 64:3932-3938 (1998); Mol. Gen. Genet. 250:734-741 (1996)) or any promoter that can be expressed in Bacillus can be used as the regulatory region.

[0063] Meanwhile, the recombinant vector of the present invention can be produced by manipulating plasmids (e.g., pCL, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8 / 9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series, and pUC19, etc.), phages (e.g., λgt4·λB, λ-Charon, λΔz1, and M13, etc.), or viruses (e.g., SV40, etc.) frequently used in the art.

[0064] Meanwhile, when the expression vector of the present invention uses a eukaryotic cell as a host, a promoter derived from the genome of a mammalian cell (e.g., metallothionine promoter, β-actin promoter, human hemoglobin promoter, and human muscle creatine promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, tk promoter of HSV, mouse mammary tumor virus (MMTV) promoter, LTR promoter of HIV, promoter of Moloney virus, promoter of Epstein-Barr virus (EBV), and promoter of Rous sarcoma virus (RSV)) can be used.

[0065] The recombinant vector of the present invention may be fused with other sequences to facilitate the purification of antibodies expressed therefrom. Examples of such fusion sequences include glutathione S-transferase (Pharmacia, USA); maltose binding protein (NEB, USA); FLAG (IBI, USA); tag sequences such as 6x His (hexahistidine; Quiagen, USA), Pre-S1, and c-Myc; and leader sequences such as OmpA and PelB. Furthermore, since the protein expressed by the vector of the present invention is an antibody, the expressed antibody can be easily purified using a protein A column or the like without additional sequences for purification.

[0066] Meanwhile, the recombinant vector of the present invention may include an antibiotic resistance gene commonly used in the art as a selection marker, for example, a resistance gene for ampicillin, gentamicin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin, and tetracycline.

[0067] The vector expressing the recombinant polypeptide of the present invention can be a vector system in which the light chain and heavy chain of the antibody contained in the recombinant polypeptide are simultaneously expressed from a single vector, or a system in which the light chain and heavy chain are each expressed from separate vectors. In the latter case, the two vectors are introduced into a host cell through co-transformation and targeted transformation. Co-transformation is a method in which each vector DNA encoding the light chain and the heavy chain is simultaneously introduced into a host cell, and then cells expressing both the light chain and the heavy chain are selected. Targeted transformation is a method in which cells transformed with a vector containing a light chain (or heavy chain) are selected, and the selected cells expressing the light chain are transformed again with a vector containing a heavy chain (or light chain), thereby finally selecting cells expressing both the light chain and the heavy chain.

[0068] The gene delivery system used in the present invention can be any gene delivery system used for conventional gene insertion, and includes, but is not limited to, plasmids, adenoviruses, adeno-associated viruses (AAV), retroviruses, lentiviruses, herpes simplex viruses, bathyscaphe viruses, liposomes, niosomes, and lipid nanoparticles.

[0069] According to another aspect of the present invention, the present invention provides a host cell transformed with the gene delivery vehicle of the present invention described above.

[0070] Any host cell capable of stably and continuously cloning and expressing the gene vector of the present invention can be used as a host cell known in the art, and for example, in the case of prokaryotic cells, it includes, but is not limited to, strains of the genus Bacillus such as Escherichia coli, Bacillus subtilis and Bacillus thuringiensis, Streptomyces, Pseudomonas (e.g., Pseudomonas putida), Proteus mirabilis or Staphylococcus (e.g., Staphylococcus carnosus).

[0071] Suitable eukaryotic host cells that can be used in the present invention may be, for example, fungi such as Aspergillus species, yeasts such as Pichia pastoris, Saccharomyces cerevisiae, Schizosaccharomyces, insect-derived cells, plant-derived cells, or mammalian-derived cells such as CHO (Chinese hamster ovary) cells.

[0072] Host cell culture for producing antibodies or antigen-binding fragments thereof can be performed according to appropriate media and culture conditions known in the art. This culture process can be easily adjusted and used by those skilled in the art according to the selected strain, and examples of specific culture methods are disclosed in various references (e.g., James M. Lee, Biochemical Engineering, Prentice-Hall International Editions, 138-176). Antibodies obtained by culturing host cells can be used in an unpurified state, or can be further purified to high purity using various conventional methods such as dialysis, salt precipitation, and chromatography. When using chromatography, the type and order of columns can be selected from ion exchange chromatography, size exclusion chromatography, and affinity chromatography, depending on the characteristics of the antibody, the host cell culture method, etc.

[0073] According to another aspect of the present invention, the present invention provides a composition for preventing or treating B cell lymphoma, comprising the recombinant polypeptide, nucleic acid molecule or gene delivery system of the present invention as an active ingredient.

[0074] According to another aspect of the present invention, the present invention provides a method for preventing or treating B cell lymphoma, comprising administering to a subject a recombinant polypeptide, nucleic acid molecule or gene delivery agent of the present invention as described above.

[0075] The term “prevention” as used herein means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed as having the disease or condition but is susceptible to such disease or condition.

[0076] As used herein, the term “treatment” means (a) suppressing the development of a disease, condition, or symptom; (b) alleviating a disease, condition, or symptom; or (c) eliminating a disease, condition, or symptom. When the composition of the present invention is administered to a subject, it blocks the proliferation of B-cell lymphomas that specifically express B-cell antigens, including CD20, on the surface and strongly induces direct cell death, thereby suppressing the progression of symptoms caused by B-cell lymphoma, eliminating them, or alleviating them. Therefore, the composition of the present invention may be a composition for treating these diseases on its own, or may be administered together with other pharmacological ingredients and applied as an adjuvant treatment for B-cell lymphoma. Accordingly, the terms “treatment” or “therapeutic agent” as used herein include the meaning of “adjuvant treatment” or “adjuvant treatment agent.”

[0077] As used herein, the term “administration” or “administer” refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount is formed in the body of the subject.

[0078] In the present invention, the term “therapeutically effective amount” means the content of a composition in which the pharmacological ingredient in the composition is contained in an amount sufficient to provide a therapeutic or preventive effect to a subject to whom the pharmaceutical composition of the present invention is to be administered, and includes a “prophylactically effective amount”.

[0079]

[0080] *The term “subject” as used herein includes, without limitation, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, monkey, chimpanzee, baboon, or rhesus macaque. Specifically, the subject of the present invention is a human.

[0081]

[0082] According to a specific embodiment of the present invention, the B-cell lymphoma that can be prevented or treated with the composition of the present invention is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), small lymphocytic lymphoma (SLL), and mantle cell lymphoma (MCL).

[0083]

[0084] According to another aspect of the present invention, the present invention provides a composition for preventing or treating B cell lymphoma, comprising:

[0085] (a) an antibody or an antigen-binding fragment thereof that specifically binds to a B-lymphocyte antigen or a nucleic acid molecule encoding the same; and

[0086] (b) MPZL1 (Myelin Protein Zero Like 1) protein or a nucleic acid molecule encoding the same.

[0087] According to another aspect of the present invention, the present invention provides a method for preventing or treating B cell lymphoma, comprising administering to a subject:

[0088] (a) an antibody or an antigen-binding fragment thereof that specifically binds to a B-lymphocyte antigen or a nucleic acid molecule encoding the same; and

[0089] (b) MPZL1 (Myelin Protein Zero Like 1) protein or a nucleic acid molecule encoding the same.

[0090] Since the antibodies specifically binding to B-lymphocyte antigens used in the present invention, antigen-binding fragments thereof, and nucleic acid molecules encoding them have already been described above, their descriptions are omitted to avoid excessive duplication.

[0091] According to the present invention, the inventors have for the first time elucidated the involvement of the MPZL1 protein in the direct apoptosis mechanism of antibody-induced B-cell lymphoma, and confirmed that MPZL1 overexpression significantly increases lymphoma cell death. Therefore, when MPZL1 protein (or its encoding nucleotide) is co-administered with a B-cell antigen-specific antibody, including CD20, a remarkable synergistic antitumor effect can be achieved.

[0092]

[0093] The features and advantages of the present invention are summarized as follows:

[0094] (a) The present invention provides a recombinant polypeptide that specifically binds to B cells and a composition for preventing or treating B cell lymphoma comprising the same as an active ingredient.

[0095] (b) The present invention can significantly enhance direct killing activity against tumor cells by conjugating concanavalin A, a ligand of MPZL1, to an antibody molecule that recognizes a B cell-specific antigen such as CD20.

[0096] (c) Accordingly, the recombinant polypeptide of the present invention, unlike therapeutic antibodies whose main mechanisms are antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADP), does not depend on the patient's immune system operation mechanism, and thus can be usefully used as an efficient therapeutic composition even for cancer patients with reduced immune activity.

[0097]

[0098] Figure 1 is a drawing showing the results of a cell death assay using a commercially available anti-CD20 antibody, showing the results of apoptosis analysis (Figure 1a) and the results of measuring LMP (lysosome membrane permeabilization) by flow cytometry after treating B cell lymphoma cells with various concentrations of rituximab and obinutuzumab (Figure 1b).

[0099] Figure 2 is a diagram showing the results of manufacturing an APEX2 fusion antibody for proximity labeling and verifying its function. The figures show the binding affinity of APEX2 and anti-CD20 antibody when they are fused without a linker (Figure 2a) and when they are linked via a linker (Figure 2c), and the results of the binding assay (flow cytometry) of the APEX2 fusion antibody and a negative control antibody with the LC-N97A mutation, respectively. Figure 2d is a schematic diagram of the expression construct of the fusion antibody in which APEX and anti-CD20 antibody (obinutuzumab) are linked via a GGGS x 4 linker.

[0100] Figure 3 is a drawing showing the results of manufacturing the second-generation APEX2 fusion antibody for short-range molecular labeling and verifying its function. The schematic diagram of the fusion antibody in which the heavy chain of obinutuzumab, rituximab, or trastuzumab is connected to APEX2 with a flexible linker, GGGGS x 4 (Figure 3a), the results of SDS PAGE analysis confirming the expression and purification of the APEX2 fusion antibody under reducing and non-reducing conditions (Figure 3b), and the binding ability of the APEX2 fusion antibody to CD20 (Figure 3c), cell killing ability (Figure 3d), and the change in cell killing rate over time (Figure 3e) are shown, respectively.

[0101] Figure 4 is a diagram showing the results of biotinylation optimization using APEX2 fusion antibodies. Protein biotinylation efficiency was confirmed according to the concentration of biotin phenol and H2O2 (Figure 4a), and the optimal concentrations of biotin phenol and H2O2 selected accordingly were applied to confirm the biotinylation efficiency according to changes in antibody concentration (Figure 4b) and antibody incubation time (Figure 4c).

[0102] Figure 5 is a drawing showing the results of Western blotting (Figure 5a) and silver staining (Figure 5b) showing the purification results of biotinylated proteins, respectively.

[0103] Figure 6 is a diagram illustrating the process of selecting protein candidates detected through HPLC-MS / MS. Among the proteins detected by Bruker TIMS-TOF PRO2, 14 final candidates were selected as membrane proteins that showed an expression change of 1.4 times or more.

[0104] Figure 7a shows the list of targets selected through HPLC-MS / MS analysis, and displays the fold change values ​​obtained by dividing the intensity values ​​in protein samples obtained by obinutuzumab-linker-APEX2 by the intensity values ​​of rituximab-linker-APEX2. Figure 7b is a diagram schematically illustrating the subcellular localization of proteins that showed higher intensity fold values ​​in obinutuzumab.

[0105] Figure 8 is a drawing showing a list of target genes selected to search for proteins involved in direct cell death (DCD) by obinutuzumab (Figure 8a) and the results of confirming the effect on the direct cell death mechanism by obinutuzumab by suppressing protein expression by doxycycline-induced shRNA using LMP assay (Figure 8b).

[0106] Figure 9 is a drawing showing the results of an LMP assay analyzing the viability of Raji cells by treating them with doxycycline-induced shRNA for a selected candidate target gene (Figure 9a) and the results of confirming the knock-down efficiency by shRNA using qPCR (Figure 9b), respectively.

[0107] Figure 10 shows the results of a DCD (direct cell death) assay performed using candidates that showed significant results in the previous LMP assay (Figure 10a) and the results of observing cell death according to the doxycycline treatment period for MPZL1 and HLA-E, the top two genes most affected by apoptosis compared to the control group (Figure 10b).

[0108] Figure 11 is a diagram showing the results of a CD20 binding assay analyzing the difference in CD20 expression level and binding affinity according to MPZL1 knockdown.

[0109] Figure 12 is a drawing showing the results of measuring the level of RNA expression in MPZL1 overexpressing cells through qPCR (Figure 12a) and the results of analyzing the level of each protein expression in knock-down and overexpressing cells through Wisconsin blot (Figure 12b).

[0110] Figure 13a shows the results of observing changes in cell death rates following treatment with doxycycline-induced shRNA targeting MPZL1, a ligand of concanavalin A, in Raji cells. Figures 13b and 13c show the results of comparing the direct cell death (DCD) induction efficiency of anti-CD20 antibodies according to the concentration of concanavalin A.

[0111] Figure 14 shows the expression construct (Figure 14a), schematic diagram (Figure 14b) of the concanavalin A-fused anti-CD20 antibody, and the results of SDS PAGE analysis of the purified fused antibody (Figure 14c), respectively.

[0112] Figure 15a shows a comparison of the apoptotic effects of rituximab and rituximab-concanavalin A (Rit-ConA). Figure 15b shows a comparison of the apoptotic effects of three wild-type antibodies and their fusion antibodies. Figure 15c shows a comparison of the efficacy and toxicity of the combined administration of concanvalin A and anti-CD20 antibodies and a fusion antibody in which they are fused into a single molecule. Figure 15d shows a comparison of the antigen binding affinity of three wild-type antibodies and their fusion antibodies.

[0113] Figure 16 shows the results of investigating the apoptotic effect of concanavalin A-conjugated antibodies on Raji cells (Figures 16a and 16b) and Ramos cells (Figures 16c and 16d), respectively.

[0114] Figure 17 is a schematic diagram showing various B cell-specific recombinant antibodies of the present invention conjugated with concanavalin A.

[0115]

[0116] Hereinafter, the present invention will be described in more detail through examples. These examples are intended solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples, in accordance with the gist of the present invention.

[0117]

[0118] Example

[0119] Experimental method

[0120] cell

[0121] CHO-K1 and HEK293T cells were purchased from the Korea Cell Line Bank, and the Raji cell line was donated by Professor Seung-Hwan Kim's laboratory at Chungnam National University. CHO-K1 and Raji cells were maintained in RPMI1640 supplemented with 10% FCS and 1% penicillin / streptomycin at 37°C and 5% CO2. HEK293T cells were maintained in DMEM high-glucose medium supplemented with 10% FCS and 1% penicillin / streptomycin at 37°C and 5% CO2. For cell culture, DMEM (Dulbecco's modified Eagle medium, 11995-065), RPMI 1640 (Roswell Park Memorial Institute medium, 11875-093), fetal bovine serum (FBS) (26140-079), penicillin-streptomycin (15140-122, Gibco, Life technologies™, Carlsbad, CA, USA); and trypsin-EDTA 0.05% solution (25300-062, Gibco, Life technologies™, Carlsbad, CA, USA) were used.

[0122]

[0123] Antibody cloning

[0124] The sequence of rituximab was obtained from U.S. Patent Publication No. 2015-0141620, and the sequence of obinutuzumab (GA101) was obtained from U.S. Patent Publication No. 11,110,087. Anti-CD20 antibody heavy and light chain coding nucleic acid sequences were cloned into the pLVX acceptor vector and introduced into lentivirus. The APEX2 sequence was obtained from Addgene (#49385, pcDNA3 Connexin43-GFP-APEX2).

[0125]

[0126] Lentivirus production

[0127] 8 x 10 per well in a 6 well plate 5293T cells were seeded, and 6 hours later, a total of 3 μg of target DNA and lentiviral DNA clones pMD2G and psPAX2 were co-cultured with 100 μL PBS and PEI (polyethylenimine) for 15 minutes, and then treated. After 6 hours of incubation, the medium (DMEM complete medium) was replaced, and the culture medium was collected after 48 hours and centrifuged at 1000 rcf for 5 minutes at 4°C. The supernatant was filtered through a 0.45 μm PES syringe filter, transferred to a clean tube to remove cell debris, and stored at -70°C.

[0128]

[0129] antibody production

[0130] For anti-CD20 antibody production, 5 x 10 per well in a 12-well plate 4 CHO-K1 cells were seeded. After 24 h of culture, polybrene (ant-pr-1, Invivogen, 10 mg / ml) was pretreated 3 h before infection. Cells were washed, treated with a 1:1 ratio of lentivirus-containing culture medium and fresh culture medium, and cultured for 24 h. The medium was replaced, and antibody-producing cells were selected by treatment with 10 μg / ml puromycin. After selection, cells were expanded for antibody production. The harvested cells were cultured in OEX-cell CD CHO medium (10743011, Thermofisher) in a CO2 incubator at 30°C for 2 weeks. The culture medium was collected, centrifuged at 1000 × g for 3 min at 4°C, and the supernatant was transferred to a clean tube to remove mixed cells and stored at 4°C. For the production of APEX2 fusion antibodies, 3 × 10 6HEK 293T cells were seeded in 100pi culture dishes. After 24 hours, 10 μg of APEX2 fusion antibody DNA was co-cultured with PEI in 200 μl of PBS for 15 minutes and treated. After 24 hours of incubation, the medium (DMEM complete medium) was replaced, and the culture supernatant was collected after 120 hours, centrifuged at 1,000 rcf for 5 minutes at 4°C, and then stored at 4°C.

[0131]

[0132] Antibody purification

[0133] Agarose A beads (Pierce Protein A Agarose, Thermofisher, QE218104) were added to the culture medium containing the antibody and incubated at 4°C for 24 hours. The medium was centrifuged at 2000 rpm for 5 minutes. The beads were placed on an activated column and washed three times with cold PBS. The antibody was eluted from the beads with elution buffer (75 μL of 0.1 M citric acid solution) and transferred to a tube containing 25 μL of 1 M Tris for neutralization. The eluted antibody was dialyzed twice against PBS for 6 hours and purified using Amincon. ⓡ The protein was concentrated using Ultra-15 Centrifugal Filter Units (UFC900324, Merck, 3K). Total protein was quantified using a NanoDrop™ Lite spectrophotometer.

[0134]

[0135] SDS-PAGE electrophoresis

[0136] After quantification, 3 μg of antibody was mixed with 6 x Laemmli sample buffer and separated on a pre-cast 10% SDS-PAGE gel under both reducing and non-reducing conditions. The gel was stained with Coomassie blue.

[0137]

[0138] Cell binding assay

[0139] B lymphoma Raji cells were resuspended in 50 μL PBS with 7, 21, 35, and 70 nM anti-CD20 antibodies (obinutuzumab, rituximab), anti-CD20-APEX2 fusion antibodies (obinutuzumab-APEX2, rituximab-APEX2), or control antibodies (trastuzumab, trastuzumab-APEX2) and incubated at 4°C for 30 min. After washing the cells with PBS, the cell pellets were resuspended in 50 μL PBS with anti-human Ig Fc-specific FITC-conjugated secondary antibody (109-095-008, 1:200 dilution; Jackson Laboratories) at a 1:500 dilution and incubated at 4°C for 30 min. After washing twice with PBS, a total of 10,000 cells were counted using a flow cytometer (FACS LSR II SORP system, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using FlowJo software.

[0140]

[0141] Antibody-dependent DCD assay

[0142] For the DCD assay, Raji cells from each well were resuspended in 300 μL RPMI complete medium and treated with the indicated doses of antibodies (7, 21, 35, and 70 nM) or trastuzumab (7 nM) for 6 h, and then stained with 1 μL of 1 mg / ml PI for 30 min at 37°C. PE-positive cells were used as a readout of apoptosis (% of fluorescence-exposed cells out of 10,000 total counted cells), counted by flow cytometry (FACS LSR II SORP system, BD Biosciences, NJ, USA) and analyzed with FlowJo software.

[0143]

[0144] APEX2 near-field molecular labeling optimization

[0145] 2 x 10 5The concentrations of antibodies, biotinylated phenol, and H2O2 for APEX2 proximal molecular labeling were optimized using Raji B-cell lymphoma cells. Reactions were performed in 50 μL of RPMI complete medium, and 0.5 mM biotinylated phenol and H2O2 were added to each APEX2 fusion antibody at 1, 3, 5, and 10 μg / ml for optimal antibody concentration. 5 μg / ml APEX2 fusion antibody was added for 15, 30, and 1 hour for optimal incubation time. For biotinylation, 0, 0.25, 0.5, 2.5, and 5 mM biotinylated phenol or H2O2 were added to each reagent at 0.5 mM H2O2 or biotinylated phenol to determine the efficiency of APEX2 labeling. Biotinylated proteins were analyzed by Western blotting.

[0146]

[0147] Protein biotinylation using APEX2 fusion antibodies

[0148] Based on the results of optimization of APEX2-dependent biotinylation, samples for HPLC-MS / MS were prepared. 1 x 10 7Raji cells were prepared, and each antibody (Obi-Tra-APEX2, Rit-APEX2, Tra-APEX2) was treated in each tube at 37°C for 30 minutes. During the incubation, 2 x and 1 x quenching solutions (0.5 mM MgCl2, 1 mM CaCl2, 5 mM Trolox, 10 mM sodium azide, 10 mM sodium ascorbate, PBS in distilled water) and 9.8 mM H2O2 solution in PBS were prepared. The cells were centrifuged at 1000 RCF for 5 minutes and the supernatant was removed. The cells were washed twice and resuspended in 1 ml of pre-warmed RPMI complete medium and 0.5 mM biotin phenol. After 1 minute, 0.5 mM H2O2 solution was treated in each tube, incubated for 1 minute, and then immediately 1 ml of 2x quenching solution was added to stop the biotinylation reaction. The biotinylated samples were centrifuged at 1000 RCF for 5 minutes, the supernatant was removed, and the cells were gently mixed with 1 x quenching solution. After additional centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml of RIPA buffer and quenching reagent, and incubated for 20 minutes at 4°C. The protein lysate was centrifuged at 18000 RCF for 10 minutes at 4°C, and the supernatant and biotinylated proteins were transferred to a new tube.

[0149]

[0150] Protein biotinylation using APEX2 fusion antibodies

[0151] Based on the results of optimization of APEX2-dependent biotinylation, samples for HPLC-MS / MS analysis were prepared. In a conical tube containing 1 ml of RPMI complete medium, 1 x 10 7Raji cells were prepared and each antibody (Obi-Tra-APEX2, Rit-APEX2, Tra-APEX2) was treated in each tube at 37°C for 30 minutes. During the incubation, 2 × and 1 × quenching solutions (0.5 mM MgCl2, 1 mM CaCl2, 5 mM Trolox, 10 mM sodium azide, 10 mM sodium ascorbate, PBS in distilled water) and 9.8 mM H2O2 solution in PBS were prepared. Cells were centrifuged at 1000 RCF for 5 minutes to remove the supernatant, washed twice, and resuspended in 1 ml of prewarmed RPMI complete medium with 0.5 mM biotinylated phenol. After 1 minute, 0.5 mM H2O2 solution was treated in each tube and incubated for 1 minute. The biotinylation reaction was terminated by immediately adding 1 ml of 2 × quenching solution. The biotinylated samples were centrifuged at 1000 RCF for 5 min, the supernatant was removed, and mixed with 1 ml of 1 x quenching solution. After additional centrifugation, the supernatant was discarded, and the cells were resuspended in 1 ml of RIPA buffer and quenching reagent, and incubated for 20 min at 4°C. The protein lysate was centrifuged at 18000 RCF for 10 min at 4°C, and the supernatant containing the biotinylated proteins was carefully transferred to a new tube.

[0152]

[0153] Purification and concentration of biotinylated proteins

[0154] Biotinylated proteins were enriched using streptavidin agarose beads. The supernatant containing biotinylated proteins was incubated with the beads at 4°C with end-to-end rotation. After incubation for 2 h, the tubes were centrifuged at 500 RCF for 2 min and washed with 1 ml of wash buffer (RIPA buffer containing 2 M urea dissolved in 1 M KCl, 0.1 M Na2CO3, and 10 mM Tris-HCl). The beads were washed repeatedly with 1 ml of detergent-free RIPA buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5 mM EDTA). After bead digestion for elution of biotinylated proteins was completed, the beads were incubated with 25 μL elution buffer 1 (50 mM Tris-HCl pH 7.5, 2 M urea, 5 μg / ml trypsin, 1 mM DTT) in a thermomixer at 400 RPM. After 30 min, the beads were centrifuged at 2500 RCF for 2 min at 4°C, and the supernatant was transferred to a new tube. Additionally, the beads were resuspended with 25 μL elution buffer 2 (50 mM Tris-HCl pH 7.5, 2 M urea, 5 mM iodoacetamide) and resuspended twice at 2500 G for 2 min at 4°C. Elutions 1 and 2 were mixed and incubated in a thermomixer at 32°C, 400 rpm, and light-shielded overnight to continue digestion. After 12 hours, the reaction was terminated by adding 1 μL trifluoroacetic acid.

[0155]

[0156] Western blot and silver staining

[0157] Lysate samples obtained from the same number of cells were mixed with 6 x lammeli sample buffer, and 20 μL of each sample was loaded onto an acrylamide gel to separate proteins by size through electrophoresis. The gel was transferred to a nitrocellulose (NC) membrane and blocked with TBS-T containing 5% skim milk and 0.1% Tween 20. After incubation for 1 hour at room temperature, the membrane was reacted with the primary antibody for 1 hour, and then washed three times with TBS-T for 10 minutes each. After this, the membrane was reacted with the secondary antibody for 1 hour at room temperature. After washing three times with TBS-T for 10 minutes each, the proteins were detected with ECL solution. 6 x lammeli sample buffer and 20 μL of each sample were separated on a pre-cast 10% SDS-PAGE gel under both reducing and non-reducing conditions. The gel was stained with Pierce™ Silver Stain Kit (ThermoFisher Scientific, cat. 24612).

[0158]

[0159] HPCL-MS / MS

[0160] Protein samples digested on streptavidin agarose beads were separated and transferred to new tubes for HPLC-MS / MS (Bruker, timsTOF Pro 2). For quantitative analysis, a final protein target list was selected from the analyzed data according to the following strategy (Fig. 6): 1) The signal intensity of each detected protein was normalized to the total intensity value obtained from the analysis. The list was compiled in order of decreasing intensity. 2) The fold change between the two anti-CD20 antibodies was calculated by dividing the normalized intensities of obinutuzumab-APEX2 and rituximab-APEX2. 3) Proteins detected by more than two unique peptides were selected. Protein lists overlapping with the control-APEX2 were excluded. 4) Proteins showing a change greater than 1.4-fold were selected. 5) Plasma membrane proteins were selected as the final protein targets.

[0161]

[0162] Development of a doxycycline-induced shRNA-dependent knockdown system

[0163] A doxycycline-induced shRNA-dependent knockdown system was developed by cloning a target shRNA antisense guide into a lentiviral vector. The UTR-targeting antisense guide RNA sequence obtained from the SplashRNA website (http: / splashrna.mskcc.org) was amplified by PCR. The amplified product was cloned into XhoI / EcoRI in the LT3GEPIR plasmid with the pRRL backbone (Addgene, Plasmid #111177). For lentivirus production, the cloned vector was co-cultured with psPAX2 and pMD2G at a concentration of 8 × 10 5 293T cells were transfected. The obtained lentivirus was 5 x 10 4 Raji cells were transduced and selected with 5 μg / ml puromycin. After selection, 1 μg / ml doxycycline was added to 1 × 10 6 Raji cells were treated and GFP-positive cells were isolated using FACS Aria Ⅲ (BD Biosciences, NJ, USA).

[0164]

[0165] Measurement of lysosomal membrane permeabilization

[0166] For LMP measurements, 1 x 10 per well 5 Raji cells were prepared and resuspended in 100 μL RPMI complete medium with 7 or 21 nM obinutuzumab or trastuzumab. After 4 h of incubation, cells were stained with LysoTracker™ Deep Red (Invitrogen™, cat. L12492) for 30 min at 37°C. The geometric mean change in LysoTracker was calculated by flow cytometry (FACS LSR II SORP system, BD Biosciences, NJ, USA) and analyzed with FlowJo software.

[0167]

[0168] RNA extraction and qPCR for gene expression quantification

[0169] A total of 3 x 10 were treated with 0.1 μg / ml doxycycline for 3 days. 6 Raji cells were prepared by inducing shRNA in dogs. RNA was isolated by suspending the cells in 500 μL Trizol and incubating them at room temperature for 5 minutes to separate nuclear proteins. After incubation, 100 μL chloroform was added, vigorously vortexed, and incubated at room temperature. After 3 minutes, the sample was centrifuged at 4°C and 13,000 RCF for 15 minutes, and the colorless liquid supernatant was transferred to a new tube. To precipitate RNA, 250 μL isopropanol was added and incubated at room temperature for 10 minutes. The solution was centrifuged at 13,000 RCF for 10 minutes at 4°C, and the RNA pellet was washed twice with 500 μL of cold 75% ethanol. The RNA pellet was centrifuged at 7,500 RCF for 5 minutes and vacuum-dried for 5 minutes to remove any residual ethanol. The RNA pellet was dissolved in 50 μL of distilled water and quantified using a NanoDrop™ Lite spectrophotometer.

[0170] The extracted RNA was reverse transcribed using the SuperiorScript III cDNA Synthesis Kit (Enzynomics, cat. EZ405S), and the obtained cDNA was used for PCR amplification using the QuantStudio 3 Real-time PCR instrument (Applied Biosystems) and AccuPower ⓡ Knockdown efficiency was measured using 2x Greenstar qPCR master mix (Bioneer, K-6251). PCR conditions were as follows: 95°C, 2 min (initial denaturation) / 95°C, 10 s; 58°C, 30 s (denaturation, annealing, extension, and measurement). Data were normalized by calculating the △△CT value between GABDH and the target gene.

[0171]

[0172] FLAG overexpression cloning

[0173] For target protein overexpression, the target gene was obtained from a transcript mutant (NCBI Gene, http: / www.ncbi.nlm.nih.gov / gene / ). The target gene, with a 3 x FLAG tag fused to the C-terminus, was cloned into the pLVX acceptor vector. Lentivirus was produced in 293T cells and introduced into doxycycline-induced knockdown Raji cells. Selection was performed by treatment with 10 μg / ml blasticidin for 5 days, and protein overexpression was confirmed by Western blotting using an anti-FLAG antibody.

[0174]

[0175] Experimental results

[0176] Antibody-dependent DCD assay and LMP assay

[0177] As a result of conducting a cell death assay using the directly produced obinutuzumab and rituximab, it was confirmed that direct cell death (DCD) occurred only with obinutuzumab (Fig. 1a), and the result of the lysosome membrane permeabilization (LMP) assay confirmed through LysoTracker™ staining that lysosomal destruction was induced only by obinutuzumab (Fig. 1b).

[0178]

[0179] Validation of APEX2 fusion antibodies for near-field molecular labeling

[0180] Fusion antibodies conjugated to the heavy chains of rituximab, obinutuzumab, and a negative control antibody (CD20-binding ability lost due to LC-N97A mutation) with APEX2 were cloned into the pLVX receptor vector, produced by transient transfection in HEK 293T cells, and confirmed by SDS PAGE (Fig. 2a). CD20 binding assays of the produced APEX2 fusion antibodies and the negative control antibody were performed by flow cytometry. The results showed that the APEX2 fusion antibodies had lower binding affinity than the wild-type antibody, and the negative control antibody showed nonspecific binding (Fig. 2b). To improve these results, a GGGS x 4 linker was introduced between the heavy chain termini of the antibodies and APEX2 (Fig. 2d), and it was observed that the binding affinity for CD20 was equivalent to that of the wild type under non-reducing conditions (Fig. 2c).

[0181]

[0182] Validation of a second-generation APEX2 fusion antibody for near-field molecular labeling

[0183] A fusion antibody was produced in which each heavy chain of a chimeric antibody introducing the heavy chains of obinutuzumab, rituximab, and trastuzumab and the light chain of obinutuzumab was linked to APEX2 via a GGGGS x 4 linker (Fig. 3a). Expression and purification of each antibody were confirmed through SDS staining under non-reducing and reducing conditions (Fig. 3b), and binding assays (Fig. 3c) and apoptosis assays (Fig. 3d and Fig. 3e) were performed on Raji cells using 35 nM of anti-CD20 antibodies (obinutuzumab, rituximab), anti-CD20-APEX2 fusion antibodies (obinutuzumab-APEX2, rituximab-APEX2), and control antibodies (trastuzumab, trastuzumab-APEX2). As a result, it was confirmed that both the binding ability to CD20 and the apoptosis ability were reduced in the case of trastuzumab (Tra) and trastuzumab-obinutuzumab-linker-APEX2 (OTA).

[0184]

[0185] Optimal biotinylation using APEX2 fusion antibodies

[0186] As a result of confirming the protein biotinylation efficiency according to the concentration of biotin phenol and H2O2, the optimal concentrations of biotin phenol and H2O2 were selected as 5 mM and 2 mM, respectively (Fig. 4a). Using these selected concentrations, the concentration of the antibody with the best biotinylation efficiency (Fig. 4b) and antibody incubation time (Fig. 4c) were selected. In addition, each biotinylated antibody (Obi-Tra-APEX2, Rit-APEX2, Tra-APEX2) was confirmed through Western blot (Fig. 5a) and silver staining (Fig. 5b) performed using the method described above.

[0187]

[0188] Selection of target proteins based on HPLC-MS / MS analysis results

[0189] The list of target proteins (genes) predicted to be involved in DCD selected according to the criteria described above (Fig. 6) through HPLC-MS / MS analysis is shown in Fig. 7a, and the fold change value obtained by dividing the intensity value in the protein sample obtained by obinutuzumab-linker-APEX2 by the intensity value of rituximab-linker-APEX2 is displayed. In addition, the effect of proteins knocked down by doxycycline-induced shRNA on cell death was analyzed by LMP assay, and no difference was observed between Raji wild type and control shLuc, and positive control shCD20 doxycycline(-), and only shCD20 doxycycline(+) ​​showed a decrease in obinutuzumab-induced cell death, confirming that the doxycycline-induced shRNA-dependent knockdown system worked well (Fig. 8b). In addition, LMP by obinutuzumab in the doxycycline-induced shRNA-dependent knockdown system was confirmed by Lysotracker Deep Red staining (Fig. 9a), and knockdown was verified by qPCR (Fig. 9b).

[0190]

[0191] Selection of DCD-involved proteins

[0192] The direct cell death (DCD) assay was performed with the candidates that showed significant results in the previous LMP assay (Fig. 10a). The effects on cell death of MPZL1 and HLA-E, two target proteins most affected by cell death compared to the control group, were examined according to the doxycycline treatment period (days 6, 9, 12, and 15) (Fig. 10b). To perform knockdown by inducing shRNA by doxycycline treatment, 2 x 10 cells were treated with 0.1 μg / ml doxycycline on days 1 and 4. 5 Cells were treated in 3 ml. Each cell was 1 x 10 5 After treating cells with 7 nM of obinutuzumab antibody for 6 hours, PI staining was performed for 30 minutes. The PI positive % was calculated using a flow cytometer, and the fold change was calculated by dividing each PI positive % by the value of cells (-) that were not treated with doxycycline.

[0193]

[0194] Confirmation of the effect of overexpression of candidate proteins

[0195] We applied the CRISPRa overexpression system to the two selected candidate proteins to determine their effect on cell death. To overexpress the target gene using the CRISPR activation system, the sequence corresponding to the guide RNA was designed and synthesized on the SplashRNA website (http: / splashrna.mskcc.org / ) and cloned into a lentiviral vector. The transcription factors MCP, P65, and HSF1 corresponding to SAM were also cloned into lentiviral vectors, and each gene vector was transfected into 293T cells. Overexpressing cells were then generated by transducing Raji cells with SAM and dCas9 lentiviruses. Stable cell lines were then established through selection with 10 μg / ml blasticidin. After RNA extraction and cDNA synthesis, overexpression was confirmed by qPCR (Figure 12a). Overexpression of the target proteins was verified by Western blotting using an anti-MPZL1 antibody. Specifically, 1 x 10 knockdown and overexpressing cells on day 6 6 After dissolving the cells in 500 μl of RIPA buffer, electrophoresis was performed on 30 μl of each sample. Detection was performed using a 1:1000 dilution of anti-human-MPZL1 mouse antibody as the primary antibody and a 1:5000 dilution of anti-mouse HRP antibody as the secondary antibody (Fig. 12b). As a result of performing an apoptosis assay using the constructed MPZL1-overexpressing Raji cells, it was confirmed that apoptosis was significantly increased by MPZL1 overexpression (Fig. 12c).

[0196]

[0197] Effects of Concanavalin A, a ligand of MPZL1, on cell death

[0198] 1 x 10 5To observe changes in the degree of cell death in Raji cells knocked down with doxycycline-induced shRNA, concanavalin A, a ligand for MPZL1, was treated at 10 μg / ml for 6 hours. 1 μL of 1 mg / ml PI was added, and staining was performed at 37°C for 30 minutes. The number of PE-positive cells was counted and expressed as the percentage of expressing cells out of a total of 10,000 cells. After calculation using a flow cytometer (FACS LSR II SORP system, BD Biosciences, Franklin Lakes, NJ, USA), the results were analyzed with FlowJo software, and it was confirmed that cell death was significantly reduced in MPZL1 knockdown cells (Fig. 13a). In addition, when direct cell death (DCD) was confirmed according to the concentration of concanavalin A and anti-CD20 antibodies (rituximab, obinutuzumab), it was observed that a synergistic cell death effect was exhibited when concanavalin A (0, 0.3, 1, 3 μg / ml) and each antibody (0, 0.3, 1 μg / ml) were treated (Fig. 13b).

[0199]

[0200] Production of concanavalin A fusion antibodies

[0201] Since it was confirmed that cell death increased when concanavalin A and anti-CD20 antibody were combined, a fusion antibody was prepared in which concanavalin A was conjugated to the anti-CD20 antibody Fc portion through the GGGGS x 4 linker used for APEX2 fusion (Fig. 14a and 14b), and the purified antibody was confirmed by SDS PAGE (Fig. 14c). The fusion antibody was cultured in HEK 293T cells at 3.5 x 10 6After seeding 100pi, transient transfection was performed with 10 μg target DNA at 1 μg DNA: 3 μl PEI. After 6 hours, the medium was exchanged, and after 24 hours of rescue, the cells were replaced with 10 ml of 293 Freestyle media and cultured for 6 days. After discarding all the supernatant, the culture medium containing the antibody was subjected to binding reaction with agarose A beads (Pierce Protein A Agarose, Thermofisher, QE218104) at 4°C for 24 hours. After centrifugation at 2000 rpm for 5 minutes, the beads were collected on an activated column and washed three times with cold PBS. The antibody was leached from the beads using a leaching solution (75 μL of 0.1 M citric acid), and 25 μL of 1 M Tris was added for neutralization. The leached antibody was dialyzed twice in PBS for 6 hours, and then resuspended in Amincon ⓡ The protein was concentrated using an Ultra-15 centrifugal filter (UFC900324, Merck, 3K). The total protein amount was measured using a NanoDrop™ Lite spectrophotometer.

[0202]

[0203] Confirming the effectiveness of concanavalin A fusion antibodies

[0204] To compare the efficacy of rituximab-concanavalin A (Rit-ConA) and rituximab, 1 x 10 5After treating Raji cells with 2.1 nM and 7 nM antibodies for 6 hours, the cell death rate was measured through PI staining, and it was confirmed that Rit-ConA showed a significantly superior cell killing effect (Fig. 15a). In addition, the result of comparing the LMP effect also showed that the concanavalin A fused antibody induced cell death more effectively than the wild-type antibody (Fig. 15b). In addition, it was confirmed that the antibody fused in a single molecule form exhibited higher cytotoxicity than when concanavalin A and anti-CD20 antibody were simply co-administered (Fig. 15c). In the case of concanavalin A, since two monomers are conjugated to the antibody, the analysis was conducted with the antibody: concanavalin A = 1:2 when co-administered.

[0205] Finally, to determine the CD20 binding affinity of each antibody, 2 x 10 6 Raji cells were resuspended in 50 μL of PBS containing 21 nM anti-CD20 antibody (obinutuzumab, rituximab), anti-CD20-conA fusion antibody, or control antibody (trastuzumab, trastuzumab-conA) and incubated at 4°C for 30 min. After washing the cells with PBS, they were resuspended in 50 μL of PBS containing anti-human Ig Fc-specific FITC-conjugated secondary antibody (109-095-008, 1:200 dilution; Jackson Laboratories) at a 1:500 dilution and incubated at 4°C for 30 min. After washing twice with PBS, a total of 10,000 cells were detected by flow cytometry (FACS LSR II SORP system, BD Biosciences, Franklin Lakes, NJ, USA) and analyzed with FlowJo software. As a result of the analysis, the binding affinity of the conA fusion antibody was slightly reduced compared to the unconjugated wild-type antibody (Fig. 15d).

[0206] Additionally, 1 x 10 5After treating Raji cells with various doses of each antibody, PI staining and Lysotracker Green staining were performed 6 hours later. As a result, it was found that the cell death of the conA fusion antibody was superior to that of the control group (Fig. 16a), which was 1 x 10 5 A similar result was observed in experiments using Ramos cells (Fig. 16b).

[0207] Amino acid sequence of antibody and concanavalin A monomer used in the present invention Sequence number Peptide sequence 1 Obi-HCDR 1 YSWIN 2 Obi-HCDR 2 RIFPGDGDTDYNGKFK 3 Obi-HCDR 3 NVFDGYWLVY 4 Obi-LCDR 1 RSSKSLLHSNGITYLY 5 Obi-LCDR 2 QMSNLVS 6 Obi-LCDR 3 AQNLELPYT 7 Obi-heavy chain variable region QVQLVQSGAEVKKPGSSVKVSCKASGYAFSYSWINWVRQAPGQGLE WMGRIFPGDGDTDYNGKFKGRVTITADKSTSTAYMELSSLRSEDTAVYYCARNVFDGYWLVYWGQGTLVTVSS8Obi-Light Chain Variable RegionDIVMTQTPLSLP VTPGEPASISCRSSKSLLHSNGITYLYWYLQKPGQSPQLLIYQMSNLVSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCAQNLELPYTFGGGTKV EIK9Rit-HCDR1SYNMH10Rit-HCDR2AIYPGNGDTSYNQKFKG11Rit-HCDR3STYYGGDWYFNV12Rit-LCDR1RASSSVSYIH13Rit- LCDR2ATSNLAS14Rit-LCDR3QQWTSNPPT15Rit-Heavy chain variable regionQVQLQQPGAELVKPGASVKMSCKASGYTFTSYNMHWVKQTPGRGLEWIGAIY PGNGDTSYNQKFKGKATLTADKSSSTAYMQLSSLTSEDSAVYYCARSTYYGGDWYFNVWGAGTTVTVASA16Rit-Light Chain Variable RegionQIVLSQSPAILSAS PGEKVTMTCRASSSVSYIHWFQQKPGSSPKPWIYATSNLASGVPVRFSGSGSGTSYSLTISRVEAEDAATYYCQQWTSNPPTFGGGTKLEIK17ConAmonomerMADTIVAVELDTYPNTDIGDPSYPHIGIDIKSVRSKKTAKWNMQNGKVGTAHIIYNSVDKRLSAVVSYPNADSATVSYDVDLDNVLPEWVRVGLSASTGLYKETNTILSWSFTSK LKSNSTHETNALHFMFNQFSKDQKDLILQGDATTGTDGNLELTRVSSNGSPQGSSVGRALFYAPVHIWESSAVVASFEATFTFLIKSPDSHPADGIAFFISNIDSSIPSGSTGRLLGLFPDAN

[0208] While specific aspects of the present invention have been described in detail above, it should be apparent to those skilled in the art that these specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant polypeptide that specifically binds to B cells comprising: (a) an antibody or an antigen-binding fragment thereof that specifically binds to a B-lymphocyte antigen; and (b) Concanavalin A protein bound to (a) above or a functional fragment thereof.

2. A recombinant polypeptide according to claim 1, characterized in that the B cell-specific antigen is at least one antigen selected from the group consisting of CD19, CD20, CD30, and CD79b.

3. A recombinant polypeptide according to claim 1, characterized in that the concanavalin A protein or a functional fragment thereof binds to the C-terminus of the antibody or an antigen-binding fragment thereof.

4. A recombinant polypeptide characterized in that in claim 3, the concanavalin A protein or a functional fragment thereof is linked to the C-terminus of the antibody or an antigen-binding fragment thereof through a linker.

5. A recombinant polypeptide, characterized in that in claim 4, the linker is a flexible peptide linker represented by (GGGGS)n (n is an integer from 1 to 5).

6. A recombinant peptide according to claim 1, characterized in that the functional fragment of concanavalin A is a dimer in which two monomer subunits constituting a concanavalin A tetramer are combined.

7. A recombinant peptide according to claim 6, characterized in that the two monomer subunits of concanavalin A bind to each heavy chain C-terminus of an antibody that specifically binds to the B cell antigen.

8. A recombinant polypeptide according to claim 2, characterized in that the B cell-specific antigen is CD20.

9. A recombinant polypeptide according to claim 8, characterized in that the antibody specifically binding to the B cell-specific antigen is obinutuzumab or rituximab.

10. A nucleic acid molecule encoding the recombinant polypeptide of claim 1.

11. A gene vector comprising a nucleic acid molecule of claim 10.

12. A host cell transformed with the gene vector of claim 11.

13. A composition for preventing or treating B cell lymphoma, comprising the recombinant polypeptide of claim 1, the nucleic acid molecule of claim 10, or the gene vector of claim 11 as an active ingredient.

14. A composition according to claim 13, wherein the B-cell lymphoma is selected from the group consisting of diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, marginal zone B-cell lymphoma (MZL), small lymphocytic lymphoma (SLL), and mantle cell lymphoma (MCL).

15. A composition for preventing or treating B cell lymphoma, comprising: (a) an antibody or an antigen-binding fragment thereof that specifically binds to a B-lymphocyte antigen or a nucleic acid molecule encoding the same; and (b) MPZL1 (Myelin Protein Zero Like 1) protein or a nucleic acid molecule encoding the same.

Images